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  • #16
    I'll e-mail it now. If you don't see it check your spam folder. E-mails from Greece tend to go there. Essentially I follow this protocol with some modifications:


    Another good thing about micrococcal nuclease fragmentation is you don't end up with a small but significant amount of higher molecular weight fragments. Also it's easier on epitopes that may be subject to stripping from the chromatin.
    --------------
    Ethan

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    • #17
      Hi all,

      ETHANol,

      I am also using the epigenome protocol for native chromatin with micrococcal digestion but don't know how to get to highest amount of chromatin together with only mononucleosomal bands.

      By any chance, could you send me your protocol as well, please?

      (I can pass you my email address)

      Thanks in advance

      Alex

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      • #18
        Hi Alex,
        First, I'm not doing native ChIP, although I have used the Epigenome-NOE native ChIP protocol and it works. I'm doing X-linked ChIP and fragmenting with micrococcal nuclease.

        I'm posting two protocols as .pdf files. The first protocol "ChIP_Protocol.pdf", I've used for ChIP-seq and it works great. The inputs are very even and don't show any bias towards open chromatin which was a problem I had with sonication. There is a clear bias to GC rich DNA but I think that comes from the library amplification. I get between 1 and 40ng of DNA depending of the antibody/antigen. Another nice thing about enzymatic fragmentation is that you do not have to dilute your samples 10 fold after sonication to get the SDS levels down to 0.1%.

        The second protocol "Quick_ChIP.pdf" I've only used for ChIP-qPCR. It works as well or better then the longer protocol. I haven't check what inputs look like when sequenced using this protocol although I am curious.

        The protocols are more like some notes I wrote to give to my student that works at the next bench, not something ready distribute to beyond so be on the look out for obvious mistakes, I have been know to be a little dyslexic.

        If anyone has any opinions on these protocols I'd like to know.
        Attached Files
        --------------
        Ethan

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        • #19
          Thanks Ethan for sharing.

          I will keep you posted with my own optimizations.

          Cheers

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          • #20
            Originally posted by ETHANol View Post
            I'd be really interested in knowing the details of your chromatin prep protocol.
            OK, the protocol is rather simple actually. Starting with ~10^8 cells I gently lyse them in 4% lysis buffer (4% SDS, 50 mM Tris, pH 8, 0.1 M NaCl, 1 mM EDTA, 0.5 mM EGTA) then carefully layer the lysate onto a cushion of 8M urea and spin the sample at 55,000 RPM for 7 hours or at 45,000 RPM overnight in a Beckman ultra-centrifuge. As I mentioned before, all the uncrosslinked DNA and proteins, and RNA remains behind in the urea. At the bottom of the tube will be a pellet that has the looks and consistency of a soft contact lens that is pure crosslinked material. The next step is to dialyze out any remaining urea. Usually I'll just wash it several times in 2 mL (the volume of the tubes I use) of PBS. You can also leave it stored in PBS until needed, this pellet will not dissolve or disaggregate. Once the pellet is cleaned we've found it works best to freeze it in liquid nitrogen and then either grind it up with a pellet pestle or pass it through an insulin syringe (this fragments the pellet into really tiny chunks that are more easily sonicated in the next step). Then I sonicate the sample in a Bioruptor or Covaris using optimized conditions for the type of chromatin being sonicated. At this point it's really no different than any other type of ChIP.

            N.B. if you were to use this method you would need to optimize the crosslinking conditions first, which you should have done anyway - if you overcrosslink the cells you won't lyse anything and you'll just have a loose clump of cells after the spin. Also, these centrifuge tubes hold ~2.2 mL of liquid. In our protocol the 10^8 cells are lysed in 800 uL of lysis buffer of which 200 uL is layered onto four tubes each containing 1.8 mL of 8 M urea.

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            • #21
              captainentropy,

              thanks for sharing your protocol. I am getting a lot of background signal from both my input and no antibody controls. So I am very interested in giving this a try.

              Wonko

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              • #22
                I don't think this will decrease your background signal. It will improve signal because there is no unbound DNA, RNA, or proteins to interefere with your ChIP or give you a false positive signal. The background problems are usually due to insufficiently blocked beads or the chromatin in the ChIP being too concentrated. Dirty antibodies and not washing well will also give high background. I occassionally still get background with this method.

                But I would like to know your results if you try it.

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                • #23
                  Hi everyone.
                  I am a new one in ChIP-seq and planning to do it for a poorly characterized TF.
                  We are going to do ChIP on transfected HEK293 cells with WT and 2 other mutant forms of our specific gene which makes three samples to be sequenced.
                  Regarding the negative control "Input DNA", the amount of samples for NGS would be 6 samples (1 sample+ its corresponding input DNA) which is quite expensive for us.
                  As I am searching to find a way to reduce the cost, I was wondering that if its possible to find the sequencing RAW files of control DNA for HEK cell somewhere in databases and use it as a control to normalize the analysis. Is it possible?
                  Are there such kind of data available and if so, can we use them instead of input DNA?
                  As I understood the sonication is not a truly random process and we may have to sequence each samples's input DNA separately.Am I correct?
                  If so, can we sequence only one of the input DNAs (for example WT) as the control for all the three samples or we have to consider one seperate input DNA for each sample?

                  I am looking forward to hearing from you.

                  Comment

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